Tissue plasminogen activator (tPA) is a thrombolytic agent which has been demonstrated to be efficacious for clots which cause myocardial infarction and pulmonary embolism. The agent, however, must be administered at high doses over several hours. Major and minor bleeding occurs in several percent of the patients treated with tPA. These complications preclude its use in the large cardiovascular disease patient population with contraindications such as previous stroke or previous myocardial infarction. Our long term objective is to generate a second-generation tPA by protein engineering techniques which has substantially higher efficacy in clot lysis. This may afford an agent which can be administered in smaller doses and with less bleeding complications. We have identified fibrin affinity as a property which can be improved and would contribute to higher efficacy in clot lysis. Phase I of this project is a molecular modeling study of the Kringle regions of tPA where we will identify amino acid residues which can be modified to increase the binding affinity of fibrin analogs. In Phase II, we will generate and test these molecules in vivo.